CN111297900A - Composition rich in adipose-derived stem cell exosomes and application thereof - Google Patents
Composition rich in adipose-derived stem cell exosomes and application thereof Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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Abstract
The composition is characterized by comprising an active ingredient adipose-derived stem cell exosome and at least one auxiliary material capable of being externally used for skin, wherein the weight percentage of the active ingredient adipose-derived stem cell exosome is 0.002% -0.005%.
Description
Technical Field
The invention relates to a composition, in particular to a composition rich in adipose-derived stem cell exosomes.
Background
Adipose-derived mesenchymal stem cells (ASMCs) are firstly reported to be separated from adipose tissues in 2001, and as the ASMCs have high proliferation and self-renewal capacity like bone marrow mesenchymal stem cells (BMSCs), a large amount of ASMCs can be separated from adipose tissues and can be multi-directionally differentiated into mesodermal and non-mesodermal cells, so that the ASMCs become hot spots of application and research of adult stem cells in regenerative medicine. Is widely applied to a plurality of research fields of skin tissue regeneration, cartilage and bone tissue regeneration, wound healing and the like.
Exosomes (Exosomes) were first discovered in the process of researching reticulocytes in 1983, and as research goes into, Exosomes were discovered to be a type of cell information carrier carrying biological activity, and the prior art document, namely research on the promotion of skin wound healing by adipose stem cell-derived Exosomes (the grand silk, the master academic paper of university of science and technology in china, 2016 (5), discloses that adipose stem cell Exosomes have certain positive effects on promoting wound healing, and thus have great potential for making various skin care products. However, in the research, it is found that the activity of the exosome produced by different inducing agents in the induction culture of the adipose-derived stem cells is different, so how to optimize the prescription to provide a composition containing adipose-derived stem cell exosomes becomes a problem to be solved in the prior art.
Disclosure of Invention
In order to solve the technical problem, the technical scheme provided by the inventor is as follows:
the composition rich in adipose-derived stem cell exosomes is characterized by comprising the adipose-derived stem cell exosomes serving as an active ingredient and at least one auxiliary material capable of being externally used for skin, wherein the weight percentage of the adipose-derived stem cell exosomes serving as the active ingredient is 0.002% -0.005%, and the adipose-derived stem cell exosomes are prepared by the following method:
A) inoculating adipose-derived mesenchymal stem cells into a first culture medium, wherein the first culture medium is a basal culture medium containing a first induction culture reagent, the basal culture medium is a low-sugar DMEM (DMEM) culture medium, and the induction culture reagent is dexamethasone, vitamin C and vitamin D3And 1-methyl-3-isopropyl-xanthine (IBMX), the first induction culture reagent being present in the culture medium in an amount of: dexamethasone 1 μmol/L, vitamin C50mg/L, and vitamin D310 to 15 μmol/L, 0.2 to 0.3mmol/L of 1-methyl-3-isopropyl-xanthine; the culture inoculated with adipose-derived mesenchymal stem cells was based on 5% CO2，Culturing at 37 deg.C for 48h, removing culture medium by suction,adding a low-sugar DMEM culture medium, culturing for 24 hours, and collecting supernatant serving as supernatant A;
B) inoculating the adipose-derived mesenchymal stem cells into a second culture medium, wherein the second culture medium is a basic culture medium containing a second induction culture reagent, the second induction culture reagent is tretinoin and β -sodium glycerophosphate, the dosage of the tretinoin in the culture medium is 0.5-1 mu mol/L of tretinoin and 2-4 mmol/L of β -sodium glycerophosphate, and the culture medium inoculated with the adipose-derived mesenchymal stem cells is based on 5% CO2，Culturing at 37 deg.C for 48h, removing culture medium by suction, adding low-sugar DMEM culture medium, culturing for 24h, and collecting supernatant as supernatant B;
C) respectively extracting exosomes from the supernatant A and the supernatant B, and then combining the exosomes as active ingredients of the composition, wherein the protein mass ratio of the exosomes extracted from the supernatant A to the exosomes extracted from the supernatant B is 1:0.5 to 1.5.
The composition is rich in adipose-derived stem cell exosomes, and the protein mass ratio of exosomes extracted from supernatant A to exosomes extracted from supernatant B is 1: 0.8 to 1.2. Most preferably 1: 1.
The method for extracting exosomes in the step C) comprises the following steps:
C1) centrifuging 300g of collected culture solution supernatant for 10min, reserving the supernatant, discarding the precipitate, and removing cells in the culture solution;
C2) centrifuging 2000-3000 g of the supernatant obtained in the step C1) for 20-30 min, retaining the supernatant, discarding the precipitate, and removing cell debris;
C3) centrifuging 10000g of the supernatant obtained in the step C2) for 60-100 min, reserving the supernatant, discarding the precipitate, and removing cell debris again;
C4) step C3), centrifuging 100000g of the supernatant for 60-120 min, discarding the supernatant, and keeping the precipitate as the extracted exosome.
The composition rich in the adipose-derived stem cell exosomes is characterized in that the weight percentage of the adipose-derived stem cell exosomes serving as active ingredients is 0.2% -0.5% (by mass of protein), the auxiliary materials capable of being externally used for skin are hydroxyethyl starch 130/0.4, sodium chloride, a pH regulator, carbomer 934, egg yolk phospholipid, vitamin E and the balance of water, the weight percentage of the hydroxyethyl starch 130/0.4 is 0.3% -0.5%, the weight percentage of the sodium chloride is 0.6% -0.8%, the pH regulator is triethanolamine, the weight percentage of the egg yolk phospholipid is 0.5% -1%, the weight percentage of the vitamin E is 0.1% -0.2%, and the weight percentage of the carbomer 934 is 0.1% -0.3%.
The composition rich in adipose-derived stem cell exosomes is further preferably used, wherein the weight percentage of the adipose-derived stem cell exosomes serving as active ingredients is 0.3-0.4%, the weight percentage of the hydroxyethyl starch 130/0.4 is 0.4%, the weight percentage of the egg yolk phospholipids is 0.8-1%, the weight percentage of the vitamin E is 0.2%, and the weight percentage of the carbomer 934 is 0.2%.
The composition rich in the adipose-derived stem cell exosomes further comprises the following steps of:
1) dispersing egg yolk lecithin in water with the prescription amount of 30-40%, sequentially adding vitamin E and adipose-derived stem cell exosomes with the prescription amount, and processing the mixture into emulsion by a high-pressure homogenizer at the temperature of 35-45 ℃;
2) dissolving hydroxyethyl starch and sodium chloride in a proper amount of water to obtain a solution;
3) dispersing carbomer 934 in a proper amount of water for full swelling, then stirring and adding triethanolamine until the pH value is 7, respectively and slowly adding the emulsion obtained in the step 1) and the solution obtained in the step 2), and supplementing the balance of water to obtain the composition.
The application of the composition rich in adipose-derived stem cell exosomes in preparing anti-skin-aging drugs.
In the research, the inventor finds that when the adipose-derived stem cells are cultured by using a specific component inducing reagent, the activity of exosomes produced by the adipose-derived stem cells can be influenced, and when the exosomes obtained under different culture conditions are used in different proportions, the generation promoting effect on human fibroblasts is different, and when the two exosomes are used together in the proportion provided by the invention, the proportion of the generated type I and type III collagens is more solved according to the proportion in the normal skin of a human body (type I: type III 3:1, see the prior art document-contents and proportions of the type I and type III collagens in the normal skin and the hypertrophic scar, Shandong university report (medical edition), volume 54: 64-67 in 2016), and the proportion of the two exosomes in the composition is determined based on the above, and in animal experiments, the composition provided by the invention also shows a better effect on preventing and treating experimental animal photoaging compared with the comparative example, in addition, the composition provided by the technical scheme of the invention can still keep a good skin care effect after storage, and the preferable auxiliary material formula is shown, so that the activity of the adipose-derived stem cell exosome can be effectively preserved.
Detailed Description
The following method was used for the preparation of the culture product of adipose-derived stem cells.
The adipose-derived stem cell exosome is prepared by the following method:
A) inoculating the adipose-derived mesenchymal stem cells into a first culture medium, wherein the inoculation amount is 50%, the first culture medium is a basic culture medium containing a first induction culture reagent, the basic culture medium is a low-sugar DMEM (DMEM) culture medium, the induction culture reagent is dexamethasone, vitamin C, vitamin D3 and 1-methyl-3-isopropyl-xanthine (IBMX), and the dosage of the first induction culture reagent in the culture medium is as follows: dexamethasone 1 μmol/L, vitamin C50mg/L, vitamin D310 μmol/L, 0.2mmol/L of 1-methyl-3-isopropyl-xanthine; the culture inoculated with adipose-derived mesenchymal stem cells was based on 5% CO2，Culturing at 37 deg.C for 48h, removing culture medium by suction, adding low-sugar DMEM culture medium, culturing for 24h, and collecting supernatant as supernatant A;
B) inoculating the adipose-derived mesenchymal stem cells into a second culture medium with the inoculation amount of 50%, wherein the second culture medium is a basic culture medium containing a second induction culture reagent, the second induction culture reagent is tretinoin and β -sodium glycerophosphate, the dosage of the tretinoin in the culture medium is 0.5 mu mol/L of tretinoin and 3mmol/L of β -sodium glycerophosphate, and the culture medium inoculated with the adipose-derived mesenchymal stem cells is based on 5% CO2，Culturing at 37 deg.C for 48h, and removing by suctionAdding a low-sugar DMEM culture medium into the culture medium, culturing for 24 hours, and collecting the supernatant as a supernatant B;
C) and respectively extracting exosomes from the supernatant A and the supernatant B, and combining the exosomes of the adipose-derived stem cells as active ingredients of the composition.
The method for extracting exosomes in the step C) comprises the following steps:
C1) centrifuging 300g of collected culture solution supernatant for 10min, reserving the supernatant, discarding the precipitate, and removing cells in the culture solution;
C2) centrifuging 2000-3000 g of the supernatant obtained in the step C1) for 20-30 min, retaining the supernatant, discarding the precipitate, and removing cell debris;
C3) centrifuging 10000g of the supernatant obtained in the step C2) for 60-100 min, reserving the supernatant, discarding the precipitate, and removing cell debris again;
C4) step C3), centrifuging 100000g of the supernatant for 60-120 min, discarding the supernatant, and keeping the precipitate as the extracted exosome.
Adipose-derived mesenchymal stem cells were adult adipose-derived mesenchymal stem cells purchased from seiko (suzhou) biotechnology limited, product catalog No. (HUXMD-01001).
And (3) taking the exosome extracted from the supernatant A as the exosome A, taking the exosome extracted from the supernatant B as the exosome B, respectively carrying out protein quantification on the exosome A and the exosome B by adopting a BCA method, and calculating the using amount of the exosome according to the protein quantification value.
Pharmacological experiment-human fibroblast I, III collagen secretion influence experiment
Adding 1000 human skin fibroblasts into each well of a cell culture pore plate, adding a low-sugar DMEM culture medium, changing the medium once every 3d, adding 15 mu L of test solution into each group after the cells are attached, setting a blank control group (adopting PBS buffer solution with the same volume), measuring the content of I, III type collagen by an ELASA method after culturing for 7d, and statistically processing data by Excel, wherein the grouping and data results are shown in the following table (means +/-s, n = 10).
The experimental result shows that compared with the exosome A and the exosome B in the ratio of 1: 0.5-1.5, the exosome A and the exosome B not only have better effect of promoting collagen secretion under the same concentration, but also secrete more type III collagen, and the proportion of the type I collagen to the type III collagen is closer to 3 in normal skin of a human body: 1. thus, a composition preparation was prepared based on this result.
Composition preparation examples
The composition rich in the adipose-derived stem cell exosomes further comprises, by mass, A% (in terms of protein), which is the active ingredient, wherein the auxiliary materials for external application to the skin comprise, by mass, hydroxyethyl starch 130/0.4, sodium chloride, a pH regulator, carbomer 934, egg yolk phospholipid, vitamin E and the balance of water, the weight percentage of the hydroxyethyl starch 130/0.4 is 0.4%, the weight percentage of the sodium chloride is 0.7%, the pH regulator is triethanolamine, the weight percentage of the egg yolk phospholipid is 0.8%, the weight percentage of the vitamin E is 0.2%, and the weight percentage of the carbomer 934 is 0.3%.
The preparation method of the composition comprises the following steps:
1) dispersing egg yolk lecithin in water with the amount of 35% of the prescription amount, sequentially adding vitamin E and adipose-derived stem cell exosomes with the prescription amount, and processing the mixture into emulsion by a high-pressure homogenizer at the temperature of 35-45 ℃;
2) dissolving hydroxyethyl starch and sodium chloride in a proper amount of water to obtain a solution,
3) dispersing carbomer 934 in a proper amount of water for full swelling, then stirring and adding triethanolamine until the pH value is 7, respectively and slowly adding the emulsion obtained in the step 1) and the solution obtained in the step 2), and supplementing the balance of water to obtain the composition.
The amounts of active ingredients used in the formulation examples and comparative examples are given in the following table (unit: weight percent)
Pharmacological experiment-anti-photoaging contrast animal experiment
5-week-old female nude mice, whose backs are separated by a midline, were coated with a blank substrate on the left side, and the composition of the example or comparative example was coated on the right side of the drug-coated area (the right side of the blank group was not coated with any test article), were coated with the drug at the morning and applied with UVA (1.76J/cm) to the skin of the backs of the nude mice 1 hour after the drug application2) And UVB (0.16 Jcm)2) The irradiation was continued for 5 days while the test article was applied every night as in the morning. The expression of MMPs and collagen in the skin of nude mice was examined after the experiment was completed. The test method comprises the following steps of dividing the test method into a blank group and test groups 1-6, wherein each group comprises 10 specimens,
1) detection of collagen content and MMPs
Hydroxyproline (Hyp) is a special amino acid of collagen in the body, and accounts for about 13.4% of the collagen of mammals, and the content of the collagen can be calculated by the content of Hyp, and the calculation formula is as follows: collagen content (mug/mgprot) = Hyp content (mug/mgprot) × 7.46, cervical spondylopathy is killed after 2h of last administration, samples are respectively taken from a matrix coating area and a medicine coating area, Hyp kit is adopted to measure Hyp content, the collagen content is calculated, and the collagen content in the matrix coating area is taken as 100%, and the collagen content relative value in the medicine coating area is calculated.
MMPs (matrix metalloproteinases), especially MMP-1 and MMP-3, are the major enzymes that degrade collagen and cause skin laxity. Wherein, MMP1 kit and MMP3 kit are used for respectively detecting the contents of MMP1 and MMP3, and the relative value of the MMPs content in the drug coating area is calculated by taking the MMPs content in the matrix coating area as 100%.
The experimental data are recorded as means ± sd, and the grouping, administration and experimental results are shown in the following table (n =10, means ± s)
The experimental result shows that the content of MMPs in the skin of the experimental animal adopting the composition in the embodiment is obviously lower than that of the experimental animal adopting a blank matrix, and the content of collagen is obviously higher than that of the experimental animal adopting the blank matrix, so that the composition provided by the invention can obviously inhibit the damage of ultraviolet irradiation of simulated sunlight on the skin of the experimental animal and shows a better skin care effect. Compared with the experimental result data of the comparative example, the preferable proportion of the exosome A and the exosome B can better play the role of resisting the skin photoaging.
Claims (8)
1. The composition rich in adipose-derived stem cell exosomes is characterized by comprising the adipose-derived stem cell exosomes serving as an active ingredient and at least one auxiliary material capable of being externally used for skin, wherein the weight percentage of the adipose-derived stem cell exosomes serving as the active ingredient is 0.002% -0.005%, and the adipose-derived stem cell exosomes are prepared by the following method:
A) inoculating adipose-derived mesenchymal stem cells into a first culture medium, wherein the first culture medium is a basal culture medium containing a first induction culture reagent, the basal culture medium is a low-sugar DMEM culture medium, the induction culture reagent is dexamethasone, vitamin C, vitamin D3 and 1-methyl-3-isopropyl-xanthine (IBMX), and the dosage of the first induction culture reagent in the culture medium is as follows: dexamethasone 1 μmol/L, vitamin C50mg/L, vitamin D310 to 15 μmol/L, 0.2 to 0.3mmol/L of 1-methyl-3-isopropyl-xanthine; the culture inoculated with adipose-derived mesenchymal stem cells was based on 5% CO2Culturing at 37 deg.C for 48h, removing culture medium by suction, adding low-sugar DMEM culture medium, culturing for 24h, and collecting supernatant as supernatant A;
B) inoculating the adipose-derived mesenchymal stem cells into a second culture medium, wherein the second culture medium is a basic culture medium containing a second induction culture reagent, the second induction culture reagent is tretinoin and β -sodium glycerophosphate, the dosage of the tretinoin in the culture medium is 0.5-1 mu mol/L of tretinoin and 2-4 mmol/L of β -sodium glycerophosphate, and the culture medium inoculated with the adipose-derived mesenchymal stem cells is based on 5% CO2，Culturing at 37 deg.C for 48h, removing culture medium by suction, adding low-sugar DMEM culture medium, culturing for 24h, and collecting supernatant as supernatant B;
C) respectively extracting exosomes from the supernatant A and the supernatant B, and then combining the exosomes as active ingredients of the composition, wherein the protein mass ratio of the exosomes extracted from the supernatant A to the exosomes extracted from the supernatant B is 1:0.5 to 1.5.
2. The composition of claim 1, wherein the protein mass ratio of exosomes extracted from supernatant a to exosomes extracted from supernatant B is 1: 0.8 to 1.2.
3. The composition as claimed in claim 2, wherein the protein mass ratio of exosome extracted from supernatant A to exosome extracted from supernatant B is 1: 1.
4. The composition as claimed in any one of claims 1 to 3, wherein the method for extracting exosomes in step C) comprises the following steps:
C1) centrifuging 300g of collected culture solution supernatant for 10min, reserving the supernatant, discarding the precipitate, and removing cells in the culture solution;
C2) centrifuging 2000-3000 g of the supernatant obtained in the step C1) for 20-30 min, retaining the supernatant, discarding the precipitate, and removing cell debris;
C3) centrifuging 10000g of the supernatant obtained in the step C2) for 60-100 min, reserving the supernatant, discarding the precipitate, and removing cell debris again;
C4) step C3), centrifuging 100000g of the supernatant for 60-120 min, discarding the supernatant, and keeping the precipitate as the extracted exosome.
5. The composition as claimed in claim 4, wherein the fat stem cell exosome is 0.002-0.005% by weight of protein as an active ingredient, the auxiliary material for external application to skin is hydroxyethyl starch 130/0.4, sodium chloride, a pH regulator, carbomer 934, egg yolk phospholipid, vitamin E and the balance of water, the hydroxyethyl starch 130/0.4 is 0.3-0.5% by weight, the sodium chloride is 0.6-0.8% by weight, the pH regulator is triethanolamine, the egg yolk phospholipid is 0.5-1% by weight, the vitamin E is 0.1-0.2% by weight and the carbomer 934 is 0.1-0.3% by weight.
6. The composition of claim 5, wherein the fat stem cell exosome is preferably used as an active ingredient in an amount of 0.003-0.004 wt%, the hydroxyethyl starch 130/0.4 wt% is 0.4 wt%, the yolk phospholipid is 0.8-1 wt%, the vitamin E is 0.2 wt%, and the carbomer 934 is 0.2 wt%.
7. The composition of claim 5 or 6, wherein the composition is prepared by a method comprising the steps of:
1) dispersing egg yolk lecithin in water with the prescription amount of 30-40%, sequentially adding vitamin E and adipose-derived stem cell exosomes with the prescription amount, and processing the mixture into emulsion by a high-pressure homogenizer at the temperature of 35-45 ℃;
2) dissolving hydroxyethyl starch and sodium chloride in a proper amount of water to obtain a solution;
3) dispersing carbomer 934 in a proper amount of water for full swelling, then stirring and adding triethanolamine until the pH value is 7, respectively and slowly adding the emulsion obtained in the step 1) and the solution obtained in the step 2), and supplementing the balance of water to obtain the composition.
8. Use of the composition rich in the adipose-derived stem cell exosomes according to any one of claims 1 to 7 in the preparation of an anti-skin-aging medicament.
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